Activators of autophagic flux and phospholipase d and clearance of protein aggregates including tau and treatment of proteinopathies

ABSTRACT

The present application discloses compounds which are activators of autophagic flux and pharmaceutical compositions comprising said activators. It further discloses use of said compounds and pharmaceutical compositions in the treatment of neurodegenerative diseases, particularly proteinopathies and tauopathies such as Alzheimer&#39;s disease. It further discloses methods of enhancing autophagic flux.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present Continuation-in-Part (CIP) application claims benefit toInternational Application Serial No. PCT/US16/055561, filed Oct. 5,2016, which International Application claims benefit to U.S. ProvisionalApplication Ser. No. 62/237,342, filed Oct. 5, 2015. The entire contentsof the above applications are incorporated by reference as if recited infull herein.

FIELD OF THE DISCLOSURE

The present disclosure relates to compounds which are activators ofautophagic flux and pharmaceutical compositions comprising saidcompounds. It further relates to use of said compounds in the treatmentof neurodegenerative diseases, particularly Alzheimer's disease.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) affects approximately five million Americansand this number is predicted to triple by 2050. At present, there are notherapies to treat Alzheimer's or other related tauopathies. Whileclinical trials using immunotherapy targeting amyloid beta (Aβ) have hadlimited success, this in only subset of those afflicted with AD or otherneurodegenerative diseases. Moreover, there are no therapies targetingother proteinopathies, including tau, the other major neuropathologicalcomponent of AD. AD accounts for most of the dementias afflictingindividuals over 65 and is estimated to cost $226 billion in healthcare,long-term care, and hospice for people with AD and other dementiasannually. This extensive economic and societal burden does not accountfor lost income of many at-home primary caregivers including spouses andother family members.

Enhancing autophagy has been shown to have therapeutic potential in thetreatment of Alzheimer's disease. Autophagic flux (including the fusionof autophagosomes to lysosomes) is a novel regulator of autophagy as itleads to the clearance of protein aggregates and reversal ofpathophysiological decline. Therefore, there exists an ongoing need forpromoters of autophagic flux and the clearance of autophagosomes bearingproteinopathies.

SUMMARY OF THE INVENTION

In some embodiments, compounds including pharmaceutically acceptablesalts thereof, which are disclosed herein, are provided.

In some embodiments a pharmaceutical composition is provided comprisinga compound disclosed herein or a pharmaceutically acceptable saltthereof. In other embodiments, methods of making the compounds andpharmaceutical compositions are also provided in, e.g., the Examplesprovided below.

In some embodiments a method of treating a neurodegenerative diseasecomprising administering to a subject in need thereof an effectiveamount of a compound or pharmaceutical composition disclosed herein isprovided.

In some embodiments a method of enhancing autophagic flux is provided.This method comprises providing to a cell or a protein aggregate aneffective amount of a compound or pharmaceutical composition disclosedherein.

These and other aspects of the invention are further disclosed in thedetailed description and examples which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 is a graph showing a photodiode array (PDA) spectrum of WHYKD8 inmouse brain.

FIG. 2 shows Western blots of LC3-II levels in primary cortical neuronsfollowing a 6 hour treatment with WHYKD1 (±BafA1) or WHYKD5.

FIG. 3 shows Western blots of LC3-II, tau, and p62 levels in organotypicslice cultures following a 6 hour treatment pith WHYKD1 (top) or WHYKD3,WHYKD5, WHYKD8, WHYKD9, or WHYKD12 (bottom).

FIG. 4 is a bar graph showing the activation of phospholipase D (PLD) bythe WHYKD series compounds (10 μM), and their ability to convertphospholipids to phosphatidylethanols in the presence of ethanol.C=Control, 12=WHYKD12, 15=WHYKD15, 19=WHYKD19, 5=WHYKD5, 8=WHYKD8,Fipi=a noncompetitive inhibitor of PLD activity.

FIG. 5 is a bar graph showing the activation of phospholipase D (PLD) bythe WHYKD series compounds (1 μM), and their ability to convertphospholipids to phosphatidylethanols in the presence of ethanol.

DETAILED DESCRIPTION OF THE INVENTION

Although macroautophagy is known to be an essential degradative processwhereby autophagosomes mediate the engulfment and delivery ofcytoplasmic components into lysosomes, the lipid changes underlyingautophagosomal membrane dynamics are undetermined. The inventors havepreviously shown that PLD1, which is primarily associated with theendosomal system, partially relocalizes to the outer membrane ofautophagosome-like structures upon nutrient starvation (Dall'Armi,2010). The localization of PLD1, as well as the starvation-inducedincrease in PLD activity, are altered by wortmannin, aphosphatidylinositol 3-kinase inhibitor, suggesting PLD1 may actdownstream of Vps34. Pharmacological inhibition of PLD and geneticablation of PLD1 in mouse cells decreased the starvation-inducedexpansion of LC3-positive compartments, consistent with a role of PLD1in the regulation of autophagy. Furthermore, inhibition of PLD resultsin higher levels of tau and p62 aggregates in organotypic brain slices.These in vitro and in vivo findings establish a role for PLD1 inautophagy.

In some embodiments, a compound is provided having the formula (II):

wherein Y¹ and Y² are independently selected from the group consistingof CH and N; wherein X is selected from the group consisting of H,halide, and aryl; wherein R¹ is selected from the group consisting ofoptionally substituted thioheteroaryl, hydroxyl-substituted(2-aminoethyl)aryl, halide, optionally substituted thiocycloalkylwherein 1-3 carbon atoms of the cycloalkyl is optionally replaced with aheteroatom selected from the group consisting of O, S and N, andthioaryl, or a salt, enantiomer, racemate, mixture thereof, orcombination thereof.

In some embodiments, the compound is selected from the group consistingof:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In one embodiment the compound is:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In another embodiment the compound is:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments, a compound is provided having the formula (III):

-   -   wherein Y¹ is CH;    -   wherein Y² is N;    -   wherein X is halide;    -   wherein R¹ is selected from the group consisting of optionally        substituted thioheteroaryl, optionally substituted        (2-aminoethyl)aryl, halide, optionally substituted        thiocycloalkyl wherein 1-3 carbon atoms of the cycloalkyl is        optionally replaced with a heteroatom selected from the group        consisting of O, S and N, and thioaryl, or a salt, enantiomer,        racemate, mixture thereof, or combination thereof.

In some embodiments, the compound is selected from the group consistingof:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments, a compound is provided having the formula (IV):

wherein X is halide;wherein R¹ is selected from the group consisting of optionallysubstituted thioheteroaryl, optionally substituted (2-aminoethyl)aryl,halide, optionally substituted thiocycloalkyl wherein 1-3 carbon atomsof the cycloalkyl is optionally replaced with a heteroatom selected fromthe group consisting of O, S and N, and thioaryl, or a salt, enantiomer,racemate, mixture thereof, or combination thereof.

In some embodiments, the compound is selected from the group consistingof:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments, a compound is provided having the formula (V):

wherein X is H;wherein R¹ is selected from the group consisting of optionallysubstituted thioheteroaryl, optionally substituted (2-aminoethyl)aryl,halide, optionally substituted thiocycloalkyl wherein 1-3 carbon atomsof the cycloalkyl is optionally replaced with a heteroatom selected fromthe group consisting of O, S and N, and thioaryl, or a salt, enantiomer,racemate, mixture thereof, or combination thereof.

In some embodiments, the compound is selected from the group consistingof:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments, a compound is provided having the formula (VI):

wherein X is H;wherein R¹ is selected from the group consisting of optionallysubstituted thioheteroaryl, optionally substituted (2-aminoethyl)aryl,halide, optionally substituted thiocycloalkyl wherein 1-3 carbon atomsof the cycloalkyl is optionally replaced with a heteroatom selected fromthe group consisting of O, S and N, and thioaryl, or a salt, enantiomer,racemate, mixture thereof, or combination thereof.

In some embodiments, the compound is selected from the group consistingof:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments, a compound is provided having the formula (VII):

wherein R¹ is selected from the group consisting of optionallysubstituted thioheteroaryl, optionally substituted (2-aminoethyl)aryl,halide, optionally substituted thiocycloalkyl wherein 1-3 carbon atomsof the cycloalkyl is optionally replaced with a heteroatom selected fromthe group consisting of O, S and N, and thioaryl, or a salt, enantiomer,racemate, mixture thereof, or combination thereof.

In some embodiments, the compound is selected from the group consistingof:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments, a compound is provided having the formula (VIII):

wherein R¹ is selected from the group consisting of optionallysubstituted thioheteroaryl, optionally substituted (2-aminoethyl)aryl,halide, optionally substituted thiocycloalkyl wherein 1-3 carbon atomsof the cycloalkyl is optionally replaced with a heteroatom selected fromthe group consisting of O, S and N, and thioaryl, or a salt, enantiomer,racemate, mixture thereof, or combination thereof.

In some embodiments, the compound is selected from the group consistingof:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments, a compound is provided having the formula (IX):

wherein Y³ is CH or N;wherein R² is optionally substituted (2-aminoethyl)aryl,or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments, the compound is selected from the group consistingof:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments, a compound is provided having the formula (X):

wherein Y³ is CH;wherein R² is selected from the group consisting of optionallysubstituted thioheteroaryl, optionally substituted (2-aminoethyl)aryl,halide, optionally substituted thiocycloalkyl wherein 1-3 carbon atomsof the cycloalkyl is optionally replaced with a heteroatom selected fromthe group consisting of O, S and N, and thioaryl,or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments, the compound is selected from the group consistingof:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments, a compound is provided having the formula (XI):

wherein R² is selected from the group consisting of optionallysubstituted thioheteroaryl, optionally substituted (2-aminoethyl)aryl,halide, optionally substituted thiocycloalkyl wherein 1-3 carbon atomsof the cycloalkyl is optionally replaced with a heteroatom selected fromthe group consisting of O, S and N, and thioaryl, or a salt, enantiomer,racemate, mixture thereof, or combination thereof.

In some embodiments, the compound is selected from the group consistingof:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments, a compound is provided having the formula (XII):

wherein Y⁴ is CH or N;wherein R³ is selected from the group consisting of optionallysubstituted thioheteroaryl, optionally substituted (2-aminoethyl)aryl,halide, optionally substituted thiocycloalkyl wherein 1-3 carbon atomsof the cycloalkyl is optionally replaced with a heteroatom selected fromthe group consisting of O, S and N, and thioaryl, or a salt, enantiomer,racemate, mixture thereof, or combination thereof.

In some embodiments, the compound is selected from the group consistingof:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments, a compound is provided having the formula (XIII):

wherein R² is selected from the group consisting of optionallysubstituted thioheteroaryl, optionally substituted (2-aminoethyl)aryl,halide, optionally substituted thiocycloalkyl wherein 1-3 carbon atomsof the cycloalkyl is optionally replaced with a heteroatom selected fromthe group consisting of O, S and N, and thioaryl, or a salt, enantiomer,racemate, mixture thereof, or combination thereof.

In some embodiments, the compound is selected from the group consistingof:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments, a compound is provided having the formula (XIV):

wherein R² is selected from the group consisting of optionallysubstituted thioheteroaryl, optionally substituted (2-aminoethyl)aryl,halide, optionally substituted thiocycloalkyl wherein 1-3 carbon atomsof the cycloalkyl is optionally replaced with a heteroatom selected fromthe group consisting of O, S and N, and thioaryl, or a salt, enantiomer,racemate, mixture thereof, or combination thereof.

In some embodiments, the compound is selected from the group consistingof:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments, a compound is provided having the formula (XV):

wherein X is H or halide;wherein Z¹ is O;wherein R⁴ is selected from the group consisting of H, optionallysubstituted alkyl, Et, CF₃, optionally substituted cycloalkyl,optionally substituted aryl, optionally substituted heteroaryl, and

In some embodiments, the compound is selected from the group consistingof:

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In one embodiment the compound is

or a salt, enantiomer, racemate, mixture thereof, or combinationthereof.

In some embodiments a pharmaceutical composition is provided comprisinga compound disclosed herein or a pharmaceutically acceptable saltthereof.

In some embodiments a method of treating a neurodegenerative diseasecomprising administering to a subject in need thereof an effectiveamount of a compound or pharmaceutical composition disclosed herein isprovided. In some embodiments the neurodegenerative disease is aproteinopathy. Proteinopathies include, but are not limited to,Parkinson's disease, Alzheimer's disease, Amyotrophic Lateral Sclerosis(ALS), Huntington's disease, chronic traumatic encephalopathy (CTE),frontotemporal dementia (FTD), inclusion body myopathy (IBM), Paget'sdisease of bone (PDB), cerebral β-amyloid angiopathy, prion diseases,familial dementia, CADASIL, amyloidosis, Alexander disease,seipinopathies, type II diabetes, pulmonary alveolar proteinosis,cataracts, cystic fibrosis and sickle cell disease. In some aspects ofthis embodiment, the proteinopathy is a tauopathy. Tauopothies includebut are not limited to, Alzheimer's disease, Parkinson's disease,Huntington's disease, progressive supranuclear palsy, chronic traumaticencephalopathy (CTE), frontotemporal dementia (FTS), Lytico-Bodigdisease, subacute sclerosing panencephalitis, ganglioglioma,gangliocytoma, and argyrophilic grain disease. In a preferredembodiment, the neurodegenerative disease is Alzheimer's disease.

In some embodiments a method of enhancing autophagic flux is provided.This method comprises providing to a cell or a protein aggregate aneffective amount of a compound or pharmaceutical composition disclosedherein.

The embodiments described in this disclosure can be combined in variousways. Any aspect or feature that is described for one embodiment can beincorporated into any other embodiment mentioned in this disclosure.While various novel features of the inventive principles have beenshown, described and pointed out as applied to particular embodimentsthereof, it should be understood that various omissions andsubstitutions and changes may be made by those skilled in the artwithout departing from the spirit of this disclosure. Those skilled inthe art will appreciate that the inventive principles can be practicedin other than the described embodiments, which are presented forpurposes of illustration and not limitation.

EXAMPLES

The following examples are provided to further illustrate certainaspects of the present invention. These examples are illustrative onlyand are not intended to limit the scope of the invention in any way.

Example 1 Example Synthetic Schemes

Scheme 1 shows the synthesis of compounds of the formula:

e.g., compounds of formula (II) and formula (III).

Representative General Procedure

A 4-chloroquinazoline and thiol were stirred in anhydrous THF at roomtemperature. A base, such as triethylamine, was added. The reactionmixture was heated to 80° C. and was stirred overnight at saidtemperature, after which it was allowed to cool to room temperature. Itwas then diluted with distilled water, and the organic material wasextracted with ethyl acetate (3×). The combined organic extracts werewashed with brine (1×) and dried with anhydrous sodium sulfate. Thesolvent was evaporated in vacua, and the crude material was purifiedeither via column chromatography or prep TLC, employing either 10% MeOHin methylene chloride or 10:1 pentane:diethyl ether as the eluent.

The above procedure is representative. Other examples disclosed hereincould be made by similar techniques or other methods known in the art.

Scheme 2 shows preparation of 1-chloro-7-fluoroisoquinoline.

Scheme 3 shows the synthesis of compounds of the formula:

e.g., compounds of formula (IV), formula (V), formula (VI), formula VIand formula (VIII).

Scheme 4 shows the synthesis of compounds of the formula:

e.g., compounds of formula (XII), and formula (XIII).

Scheme 5 shows the synthesis of compounds of the formula:

e.g., compounds of formula (IX), formula (X), and formula (XI).

Scheme 6 shows the synthesis of compounds of the formula:

e.g., compounds of formula (XIV).

Example 2 Activators of Autophagic Flux and Phospholipase D

The WHYKD series of compounds were synthesized for optimal brainpenetrance based on the molecular weight (MW) and partition coefficient(log P), according to Lipinski's Rule for CNS penetrance: MW≤5. 400, logP≤5.

Activators according to the formula:

were synthesized according to the schemes above. Molecular weights andlog P were calculated. Results are shown in Table 1 below.

TABLE 1 PROJECT STRUCTURE ID M.W. log P X Y¹ Y² R¹

WHYKD3  323.17 3.85 Br N N thioheteroaryl

WHYKD4  369.44 5.69 aryl N N Thioheteroaryl

WHYKD5  262.27 3.18 F N N Thioheteroryl

WHYKD6  244.28 3.02 H N N thioheteroaryl

WHYKD7  278.72 3.58 Cl N N thioheteroaryl

WHYKD8  299.76 3.91 Cl N N (2-aminoethyl)aryl

WHYKD9  182.58 2.58 F N N Cl

WHYKD10 243.29 2.9  H N CH thioheteroaryl

WHYKD11 261.28 3.06 F N CH thioheteroaryl

WHYKD12 262.35 4.38 F N N thiocycloalkyl

WHYKD13 316.44 5.21 F N N thiocycloalkyl

WHYKD14 314.42 4.66 F N N thiocycloalkyl

WHYKD15 248.32 3.96 F N N thiocycloalkyl

WHYKD16 274.36 4.19 F N N thiocycloalkyl

WHYKD17 357.49 4.09 F N N thiocycloalkyl

WHYKD18 386.48 4.41 F N N thiocycloalkyl

WHYKD19 264.32 2.63 F N N thiocycloalkyl

WHYKD20 296.36 4.8  F N N thioaryl

WHYKD30 356.23 5.16 I N N thiocycloalkyl

Activators according to the formula:

were synthesized according to the schemes above. Molecular weights andlog P were calculated. Results are shown in Table 2 below.

TABLE 2 PROJECT STRUCTURE ID M.W. log P Y³ R²

WHYKD21 272.33 3.36 N (2-aminoethyl)aryl

WHYKD23 271.34 3.66 CH (2-aminoethy)aryl

Activators according to the formula:

were synthesized according to the schemes above. Molecular weights andlog P were calculated. Results are shown in Table 3 below.

TABLE 3 PROJECT STRUCTURE ID M.W. log P Y⁴ R³

WHYKD1  251.29 2.56 N thioheteroaryl

WHYKD2  272.33 2.89 N (2-aminoethyl)aryl

WHYKD22 271.34 3.34 CH (2-aminoethyl)aryl

Activators according to the formula:

were synthesized according to the schemes above. Molecular weights andlog P were calculated. Results are shown in Table 4 below.

TABLE 4 PROJECT log STRUCTURE ID M.W. P X Y¹ Y² R⁴ Z¹

WHYKD24 164.14 1.02 F N N H O

Example 3 Design of Derivatives

Several series of derivatives were synthesized based on the followinglead compounds:

In addition to log P, the topological polar surface area (tPSA), CLogP(log P calculated by group contribution method), and LogS (solubility)were calculated. Results are shown in the Tables below.

TABLE 5 Modifications to the core and side chain (Series 1) STRUCTURElog P tPSA CLogP LogS

3.35 52.68 2.65154 −3.235

3.12 61.47 2.34241 −3.295

2.94 40.32 1.83259 −4.663

3.19 27.96 3.25375 −3.864

4.14 12.36 4.64041 −4.354

2.71 49.11 2.01759 −4.354

2.95 36.75 3.23654 −3.554

2.8  21.59 2.80041 −3.813

4.56 12.36 5.19941 −4.832

TABLE 6 Modifications to the core and side chain (Series 2) STRUCTURElog P tPSA CLogP LogS

2.31 77.4 0.803829 −1.704

2.07 86.19 0.539011 −1.765

1.9 65.04 −0.0366305 −3.133

1.66 73.83 0.148224 −2.824

2.14 52.68 1.40054 −2.334

1.91 61.47 1.38428 −2.024

3.09 37.08 2.83701 −2.823 (WHYKD33)

3.51 37.08 3.39601 −3.301

1.76 46.31 0.997011 −2.283

TABLE 7 Modifications to the core and side chain (Series 3) STRUCTURElog P tPSA CLogP LogS

2.89 77.4  0.647513 −1.626

2.85 86.19 0.382662 −1.686

2.48 65.04 −0.192932 −3.117

2.25 73.83 −0.00808129 −2.806

2.73 52.68 1.24423 −2.303

2.49 61.47 1.22796 −1.992

3.68 37.08 2.68066 −2.893 (WHYKD35)

4.09 37.08 3.23966 −3.372

2.34 46.31 0.840662 −2.256

TABLE 8 Modifications to the core and side chain (Series 4) STRUCTURElog P tPSA CLogP LogS

1.68 77.4  0.647513 −1.441

1.45 86.19 0.382662 −1.501

1.28 65.04 −0.192932 −2.932

1.04 73.83 −0.0080129 −2.621

1.52 52.68 1.24423 −2.119

1.28 61.47 1.22796 −1.808

2.47 37.08 2.68066 −2.704

2.89 37.08 3.23966 −3.183

1.13 46.31 0.840662 −2.071

TABLE 9 Modifications to the core and side chain (Series 5) STRUCTURElog P tPSA CLogP LogS

1.68 77.4  0.647513 −1.466

1.45 86.19 0.382662 −1.526

1.28 65.04 −0.192932 −2.957

1.04 73.83 −0.00808129 −2.646

1.52 52.68 1.24423 −2.144

1.28 61.47 1.22796 −1.832

2.47 37.08 2.68066 −2.733

2.89 37.08 3.23966 −3.212

1.13 46.31 0.840662 −2.096

TABLE 10 Modifications to the core and side chain (Series 6) STRUCTURElog P tPSA CLogP LogS

2.11 77.4  0.857513  −1.525

1.87 86.19 0.592663  −1.585

1.7  65.04 0.0170677 −3.017

1.46 73.83 0.201919  −2.705

1.94 52.68 1.45423  −2.203

1.71 61.47 1.43796  −1.892

2.89 37.08 2.89066  −2.787 (WHYKD36)

3.31 37.08 3.44966  −3.266

1.55 46.31 1.05066  −2.155

TABLE 11 Modifications to the core and side chain (Series 7) STRUCTURElog P tPSA CLogP Logs

1.63 74.27 1.1098  −1.275

1.4  83.06 0.834   −1.333

1.23 61.91 0.272969 −2.704

0.99 70.7  0.457768 −2.391

1.47 49.55 1.70682  −1.904

1.24 58.34 1.69005  −1.592

2.42 33.95 3.132   −2.403

2.84 33.95 3.691   −2.883

1.08 43.18 1.292   −1.864

TABLE 12 Modifications to the core and side chain (Series 8) STRUCTURElog P tPSA CLogP LogS

1.96 74.27 0.8996   −1.745

1.72 83.06 0.624   −1.803

1.55 61.91 0.0629689 −3.174

1.31 70.7  0.247768  −2.862

1.79 49.55 1.49682  −2.374

1.56 58.34 1.48005  −2.062

2.74 33.95 2.922   −2.874

3.16 33.95 3.48    −3.353

1.4  43.18 1.082   −2.335

TABLE 13 Modifications to the core and side chain (Series 9) STRUCTURElog P tPSA CLogP LogS

3.0  65.04 1.74907  −2.051

2.76 73.83 1.47586  −2.109

2.59 52.68 0.911314 −3.542

2.36 61.47 1.09641  −3.23

2.84 40.32 2.34546  −2.728

2.6  49.11 2.32952  −2.416

3.79 24.72 3.77388  −3.323 (WHYKD32)

4.2  24.72 4.33286  −3.802

2.45 33.95 1.93386  −2.687

TABLE 14 Modifications to the core and side chain (Series 10) STRUCTURElog P tPSA CLogP LogS

2.94 65.04 1.53907  −2.188

2.71 73.83 1.26586  −2.247

2.54 52.68 0.701314 −3.68 

2.3  61.47 0.886405 −3.367

2.78 40.32 2.13546  −2.866

2.55 49.11 2.11952  −2.554

3.73 24.72 3.56386  −3.468

4.15 24.72 4.12286  −3.947

2.39 33.95 1.72386  −2.824

TABLE 15 Quinazolinones (Series 11) STRUCTURE log P tPSA CLogP LogS

1.02 41.46 0.506065 −1.702

1.42 41.46 1.07606  −2.152

1.69 41.46 1.22606  −2.273

0.86 41.46 0.305   −1.452

Example 4 Biological Testing of WHYKD Compounds

The assays listed below were carried out using a transfected HEK 293(Human Embryo Kidney) cell line that has been engineered to expressfluorescently tagged (mKate2) Tau (unless otherwise noted). The cellswere grown in the presence of the antibiotic doxycycline, When theantibiotic is removed the cells produce Tau (which can be quantified),thus allowing the test compounds' effects on Tau's production to becompared. Doxycycline was removed for 72 hours prior to exposure to thetest compounds to promote sufficient Tau production. Cells weresubsequently plated into plates for each of the assays described below.

Autophagy, Aggregate and Tau-mKate2 IC50 Assays Preparation of PlatedTet-Regulated HEK 293 Cells (Jump-In™ Cells)

1) HEK cells with tet-regulated expression of mKate2 tagged Tau weregrown in DMEM medium (Dulbecco's Modified Eagle's Medium) containing 0.5μg/mL doxycycline (Dox)(MP-Bio# 198955), after 3-5 days Dox was removedby washing the cells twice with sterile phosphate buffered saline (PBS;Invitrogen# 14190-144), cells were left in DMEM without Dox for 72 hours(to further clear remaining Dox in cells).

2) 96 well, black plates with ultra-thin clear bottom (Costar#3720) werecoated with Poly D Lysine (PDL) (Sigma—p0899—M wt. 70,000-150,000) orcommercially sourced transparent polystyrene/glass bottom plates wereused and coated. PDL was aspirated after 2 hours and plates were allowedto dry out for another 2-3 hours. The coating procedure was completedunder sterile conditions. Plates were used immediately after coating.

3) Cells were detached from the flasks using triple express(Invitrogen#12605-010) and plated in PDL-coated 96 well plates. Forplating, 200 μL of medium was added to each well at a concentration of400 k cells/ml (80 k cells/well). 1 row of peripheral wells on all sideswas spared to prevent any changes in experimental conditions due toevaporation from these wells. Warm medium/PBS (containing no cells) waspipetted into the peripheral wells and PBS was pipetted into spacesbetween wells to maintain homogenous conditions across the centralwells. Cells were then allowed to settle down and attach to the bottomof the plate for 18-24 hours.

Treatment and Staining with Cyto-ID™

Cyto-ID™ assay measures autophagic vacuoles and monitors autophagic fluxin lysosomally inhibited live cells using a novel dye that selectivelylabels accumulated autophagic vacuoles. The dye used in the kit preventsits accumulation within lysosomes, but enables labelling of vacuolesassociated with the autophagy pathway using the LC3 biomarker.

The test compounds (along with CytoID™ dye and Hoechst stain) were addedto the cells and incubated for 3 hours. Reference compounds were alsoused in each plate, Rapamycin was used for autophagy induction andchloroquine was used for lysosomal inhibition. After 3 hours testcompound was aspirated off and kept. Cells were then washed and readusing a fluorescence plate reader. Hoescht, CytoID™ and mKate2 weremeasured using 3 distinct wavelengths on the plate reader. The Hoeschtmeasurement allowed normalisation of results across wells. Afterreading, test compound was re-added and the cells are left for a further21 hours. At the 24 hour time point cells were once again washed andplates read using the fluorescence plate reader.

1) Before initiating treatment, all wells were washed once with warmFluoroBrite™ (FB) DMEM (Dulbecco's Modified Eagle's Medium) (Invitrogen#A18967-01) with 10% Fetal Bovine Serum, dialyzed (FBS; Invitrogen#26400-044) and 1% MEM (Minimal Essential Media) NEAR (Non EssentialAmino Acids). 280 μl of warm medium/PBS was maintained in peripheralwells.

2) Test samples were prepared in warm FiuoroBrite™ DMEM and with 10% FBSand 1% NEAA (FB-DMEM). Rapamycin 200 nM (Enzo #BML-A275-0025) was usedfor autophagy induction and 15 mM chloroquine was used for lysosomalinhibition (bafilomycin or similar compounds were not used as they givea false negative result in the assay). Dimethyl Sulfoxide 1:1000 (DMSO;Fisher #BP231-100) was used as control. Autophagosome marker Cyto ID™(1:500; Enzo #ENZ-51031-k200) and Hoechst (1:200) was added to thesetest sample preparations before treating the cells.

3) The treatment groups were staggered in order to obtain similarconditions across all groups. For an n=6, 3 control wells were near theperiphery and 3 were near the center of the plate, same was true forRapamycin and any other drug treatments.

4) Cells were treated/stained for 3 hours at 37° C. At 3 hourspost-treatment, test sample preparation containing Cyto-ID™ wascarefully aspirated and transferred to a fresh sterile microplate(Falcon# 353072) for reuse. Medium in the treatment plate wasimmediately replaced with warm FB-DMEM. The new plate with test compoundpreparations was placed in an incubator at 37° C.

5) Treatment plate was then washed quickly 3 times with warm FB-DMEM(100 μL/well). After the 3rd wash 50 μL of warm medium was left in eachwell. At this point, the plate was ready for reading. See details inReading Plate section. Hoescht was read at Excitation wavelength(Ex)=355nm & Emission wavelength (Em)=446nm. mKate2 was read at Ex=575nm & Em=630 nm. Cyto-ID™ was read at Ex=463 nm & Em=534 nm.

6) Afterwards, medium was replaced with corresponding wells of the platecontaining test compound/stain preparation and re-incubated at 37° C.

7) At 24 hours post reaction all medium was aspirated and the plate waswashed 3 times with warm FB-DMEM, and read again for Hoescht (Ex=355 nmEm=446 nm), CytoID™ (Ex=463 nm; Em=534 nm) and mKate2 (Ex=575 nm; Em=630nm) in 80 μl of FB-DMEM.

Proteostat™ Protein Aggregation Assay

Proteostat™ assay was used to detect aggresomes via measurement of p62.Aggresomes are inclusion bodies that form when the ubiquitin-proteasomemachinery is overwhelmed with aggregation-prone proteins. Typically, anaggresome forms in response to some cellular stress, such ashyperthermia, viral infection, or exposure to reactive oxygen species.Aggresomes may provide a cytoprotective function by sequestering thetoxic, aggregated proteins and may also facilitate their ultimateelimination from cells by autophagy. Following the final plate readdescribed in the Cyto-ID™ assay described above, Proteostat™ detectionreagent was added to every well and the plate was incubated for 15minutes. Following this incubation, the plate was read by fluorescenceplate reader at the specified wavelength. After this plate read, thecells were fixed by incubating with warm paraformaldehyde for 8 minutes.The fixed cells were then read by plate reader as before.

8) Detection solution was prepared by adding 10 μl Proteostat™ detectionreagent (ENZ-51023-KP002) and 200 μL of 10× assay buffer into 1790 μLwater and mixing well.

9) 20 μL was added per well (each well had 80 μl of Fluorobrite™ mediawith no FBS) and incubated in the dark for 15 min at room temperature.

10) Fluorescence for Proteostat™ (Ex=550 nm; Em=600 nm) was then read.

11) Plate was fixed by adding warm 4% paraformaldehyde 100 μL (PFA; EMS#15710) to all the wells and was incubated at room temperature for 8 min.PFA was removed and plate was washed with PBS (room temp) 3 times.

12) Plate was read again with fixed cells and same configuration atplate reader.

Note: CytoID™ and Proteostat™ are measures for LC3 and p62, which can besubstituted using fluorescent tagged antibodies with correspondingfluorophores.Reading Plate using Tecan M200

13) Plate was transferred to plate reader (Tecan Infinite M200) and readat optimal gain for mKate2, Cyto ID™ and Proteostat™ (last read only).

14) Each well was read at 5 consistent locations per well for the threesignals. Each of these 5 locations was flashed 25 times. Signal fromeach well was first recorded as an average of 25 flashes, and the finalvalue was based on average of 5 read locations per well. A peripheralborder of 1000 μm was spared in all wells to mitigate anyinconsistencies in reads due to minor cell loss across the peripheryresulting from aspiration and washings. Peripheral wells were used todetect any background noise due to medium or PBS.

15) mKate2 was read at Ex=575 nm and Em=630 nm. CytoID™ was read atEx=463 nm and Em=534 nm. For the final read, Proteostat™ was read atEx=490 nm and Em=600 nm. Excitation & Emission Bandwidth for all threereads were 9 nm & 20 nm respectively.

16) Calculations were initially subtracted from well background (mediaonly well) and normalized using Hoescht levels,

Reading Plate using IN Cell Analyzer 2000 (High content imaging),

17) Plate was transferred to IN Cell analyzer 2000 and imaged formKate2, Cyto ID™, Proteastat™ and Hoechst (last read only).

18) Each well was imaged at 4 consistent fields located around thecenter of the well. All images were taken using 20× objective. Theaverage reading from these 4 fields was recorded as the reading for thatcorresponding well. No images were taken from periphery of the wells tomitigate any inconsistencies in reads due to minor cell loss across theperiphery resulting from aspiration and washings. Peripheral wells wereused to detect any background noise due to medium or PBS.

19) FITC/FITC filter (Ex=490 nm−bandwidth 20 nm/Em=525 nm−bandwidth 36nm) was used to image Cyto ID™, mKate2 was imaged usingTexasRed/TexasRed filters (Ex=579 nm−bandwidth 34 nm/Em=624 nm−bandwidth40 nm). For the Proteostat™ images FITC/dsRed combination was used(Ex=490 nm−bandwidth20 nm/Em=605 nm−bandwidth 52 nm). Nuclei were imagedusing DAPI/DAPI filter set (Ex=350 nm−bandwidth 50 nm/Em=455nm−bandwidth 50 nm).

p62 Aggregate and Tau Aggregate Western Btot Assay

Western blot assays were performed to determine protein changes in Tauand p62. Cells were cultured as above and incubated with test compoundsfor 24 h. Following test compound incubation, the test compound wasaspirated off and cells were washed before harvesting. Cells were spunat a low speed and supernatant was aspirated to leave the cell pellet.The cell pellet was then homogenized in buffer, centrifuged at highspeed, and the supernatant aspirated and further separated into totalfraction and aggregate fraction allowing quantification of soluble andinsoluble proteins. Western blots were run on the samples, gelstransferred to nitrocellulose and incubated with antibodies for Tau andp62. After incubation with secondary antibodies for detection, the bandsof protein were quantified by chemiluminescence.

1) Jump-In™ cells (see above) were maintained in 0.5 μg/mL doxycyclineuntil use.

2) Three days prior to plating, cells were replated at 40% confluency inmedia without doxycycline.

3) The day prior to experimentation, 750 000 cells were plated per welln a 6-well plate (250 000 cells in 12 well plate).

4) On the day of experiment, cells were washed in warmed HBSS (Hank'sBalanced Salt Solution) twice before media containing test compound (n=3per compound per dose) or vehicle were added to the wells (1.5 mL in 6well, 600 μL in 12 well). Plates were incubated for 24 hours at 37° C.with 5% CO₂.

5) Cells were rinsed twice in warmed HBSS before harvesting in 1 mL HBSSand transferred to 1.5 mL microtubes

6) Samples were spun at 500×g for 2 min at 4° C., and HBSS supernatantaspirated, leaving only the cell pellet. The cell pellet may be flashfrozen and stored at −80° C. until use.

Sample Preparation

7) The cell pellet was homogenized in RIPA+ (Radio-ImmunoprecipitationAssay) buffer containing protease inhibitors and phosphatase inhibitorsand gently homogenized using a cell homogenizer.

8) Samples were then centrifuged for 20 min at 20,000 g at 4° C.

9) The supernatant was transferred to a new tube.

10) The supernatant was quantified for protein concentration using thePierce™ Protein Assay.

11) Total fraction: 200 μg of supernatant was used to make a 1 mg/mlprotein solution by adding RIPA+buffer to bring the volume to 130 μL andadding 20 μL of 1M DTT (dithiothreitol) (to make a final concentrationof 100 mM DTT) and 50 μL of 4× Invitrogen NuPAGE LDS (lithium dodecylsulfate) loading buffer (4×, 10 ml-NP0007) with 100 mM DTT.

12) Aggregate fraction: For aggregates, 100 μg of sample was brought upto a final volume of 900 μL.

13) 100 μL of 10% sarkosyl solution was added to the sample and rotatedat 4° C. for 60 min.

14) The sample was then centrifuged at 100,000 g for 60 min at 4° C.

15) The supernatant was carefully removed, leaving the pelletundisturbed. The tube was inverted to remove any additional liquid. Ifthere was excess liquid, steps 12-14 were repeated to ensure a pureaggregate sample. Pellets were resuspended and solubilized in 65 μL ofPBS before the addition of 10 μL of 1M and 25 μL of 4× lnvitrogen NuPAGELDS loading buffer with 100 mM OTT.

Electrophoresis/Western Blot

16) Prior to loading, the samples were heated at 90° C. for 2 min. Aquick spin of the samples was performed to ensure there was nocondensation on the tube. The 4-12% Tris-Bis gel was prepared using 1×MOPS buffer (Invitrogen—NP0001) and anti-oxidant (NP0005). For thesoluble fraction, 2 μg of sample was loaded to each well; for theinsoluble fraction, 5 μg of sample was loaded per well. 8 μL ofInvitrogen Sharp MW standard was then loaded (optimally, all wells hadthe same volume of sample buffer.)

17) Sample was electrophoresed at 150V for approximately 1 h 15 min(until the running dye reached the base of the gel.)

18) The gel was then equilibrated in transfer buffer (25 mM Tris-HCl pH8.3, 192 mM glycine, 20% (v:v) methanol) for 5 min.

19) The gel was then removed and transferred onto 0.2 μM nitrocellulose(GE BA83 10600001) at 200 mA for 90 minutes.

20) After transfer, the blot was briefly stained (15 s) with 0.1%Ponceau S in 5% acetic acid to ensure consistent sample loading betweenlanes.

21) The blot was then rinsed in TBS-T (Tris-buffered saline-polysorbate)for 2 min to remove the Ponceau S stain prior to immunoprobing.

Immunodetection

22) Samples were blocked in 5% milk in TBST for 30 min, and then rinsedin TBS-T until all buffer was clear (no milk residues remained insolution).

23) Blots were incubated in primary antibody (1:4000 PHF1 or CP27 fortau; 1:4000 p62 Abnova for p62; 1:5000 GAPDH (glyceraldehyde 3-phosphatedehydrogenase) for loading control) overnight in SuperBlock™ TBS-T at 4°C. on a rocking table.

24) After primary antibody exposure, blots were washed three times inTBS-T for 15 min.

25) Blots were then incubated in 1:4000 secondary antibody (JacksonLaboratories goat anti-mouse HRP conjugate).

26) After secondary, blots were washed three times in TBST for 15 min.

27) Blots were then developed using Millipore chemiluminescent fluid (1ml per reagent; WBKLS0500) and detected using a Fuji LAS3000 Imagingunit at increments of 10 seconds.

28) Images were quantified using resident software or NIH Image J.

PLD Assay

Cells were incubated with test compound for 24 hours. 25 minutes priorto harvest at 24 hours, 3% ethanol solution was added to the wells tocatalyze cleavage of the phospholipid. Cells were then placed on ice,washed and harvested. The harvested cells were centrifuged and thesupernatant was aspirated and the pellet kept. The pellet wasresuspended and chloroform/methanol lipid extraction was performed. Thesample was centrifuged and the organic layer was separated, dried undernitrogen and stored at −80° C. On the day of analysis, samples wereresuspended and analysed by LC/MS. All phosphatidylethanol species(PEtOH32-40:0-6:16/18:0/1) were combined together and represented astotal lipid content (all lipid species).

The assay was run in e18 primary cortical neurons from PLD1 or PLD2 KOmice cultured for 14 days. Alternatively, the assay can be run in thecells described above in the presence of excess PLD1 or PLD2 inhibitorto preclude that particular action from contributing to the effect ofthe drug.

1) Jump-In™ cells (see above) were maintained in 0.5 μg/mL doxycyclineuntil use. For neurons, e18 fetuses were used, cortical neuronsextracted and plated onto PLD-collagen coated 6 well plates andincubated for 14 days prior to use.

2) Three days prior to plating, cells were replated at 40% confluency inmedia without doxycycline.

3) The day prior to experimentation, 750 000 cells were plated per wellin a 6-well plate (250 000 cells in 12 well plate).

4) On the day of experiment, cells were washed in warmed HBSS twicebefore media containing test compound (n=3 per compound per dose),inhibitor (355 nM ML298 or 50 nM VU0150669), or vehicle are added to thewells (1.5 mL in 6 well, 600 μL in 12 well). Plates were incubated for24 hours at 37° C. with 5% CO₂.

5) 25 minutes prior to harvest, a 165 μL of a 3% ethanol solution wasadded to each well.

6) For harvesting, plates were placed on ice and cells were rinsed twicewith ice cold HBSS before harvesting in 1 mL HBSS and transferring to1.5 mL microtubes.

7) Samples were spun at 500×g for 2 min at 4° C., and HBSS supernatantaspirated, leaving only the cell pellet. The cell pellet could be flashfrozen and stored at −80° C. until use.

TABLE 16 Biological Testing of WHKD Compounds 1-24 % Inhibition %Increase Cytotoxixity Compound Tau/aggregate (1 μM) Autophagy MarkersPLD LC50 WHYKD1 No Effect @ 2 μM No Effect @ 2 μM NC >100 μM WHYKD2 NoEffect @ 2 μM No Effect @ 2 μM ND >100 μM WHYKD3 No Effect @ 2 μM NoEffect @ 2 μM NC >100 μM WHYKD4 No Effect @ 2 μM No Effect @ 2 μMND >100 μM WHYKD5 35% 20%   1.7X >100 μM WHYKD6 No Effect @ 2 μM NoEffect @ 2 μM ND >100 μM WHYKD7 No Effect @ 2 μM No Effect @ 2 μMND >100 μM WHYKD8 30% 25%   2.8X >100 μM WHYKD9 No Effect @ 2 μM NoEffect @ 2 μM ND >100 μM WHYKD10 No Effect @ 2 μM No Effect @ 2 μMND >100 μM WHYKD11 No Effect @ 2 μM No Effect @ 2 μM ND >100 μM WHYKD1260% 40% 3.4 >100 μM WHYKD13 No Effect @ 2 μM No Effect @ 2 μM ND >100 μMWHYKD14 No Effect @ 2 μM No Effect @ 2 μM ND >100 μM WHYKD15 65% 40%3.6 >100 μM WHYKD16 No Effect @ 2 μM No Effect @ 2 μM ND >100 μM WHYKD17No Effect @ 2 μM No Effect @ 2 μM ND >100 μM WHYKD18 No Effect @ 2 μM NoEffect @ 2 μM ND >100 μM WHYKD19 55% 35% 3.5 >100 μM WHYKD20 No Effect @2 μM No Effect @ 2 μM ND >100 μM WHYKD21 No Effect @ 2 μM No Effect @ 2μM ND >100 μM WHYKD22 No Effect @ 2 μM No Effect @ 2 μM ND >100 μMWHYKD23 No Effect @ 2 μM No Effect @ 2 μM ND >100 μM WHYKD24 30% 15%ND >100 μM ND—not done, NC—no change

TABLE 17 Biological Testing of WHKD Compounds 30-36 EC50 (nM) - EC50(nM) - TI - Compound LC50 (mM) Proteostat mKate2 mKate2 WHYKD30 >2002510 1113 180 WHYKD32 >200 1992 1562 128 WHYKD33 >200 8805 637 314WHYKD35 >200 1295 518 386 WHYKD36 67 383 1044 64LC50 (500/ lethal concentration) was tested using an XTT assay for cellviability. Measurements listed as 200 represent the upper concentrationlimit used for testing, whereby the viability is >50% at this upperlimit.EC50 (50% reduction) was based on the concentration whereby the levelswere 50% lower than the initial readings based on mKate2 fluorescence(tau tag) or Proteostat levels (fluorescent marker of aggregates).TI (therapeutic index) was based on the ratio of LC50:EC50, with theupper limit being 200 mM

TABLE 18 PLD1 & PLD2 Activity Proteostat (nM) using mKate2 (nM) usingpharmacological inhibitor pharmacological inhibitor Compound PLD1 PLD2PID2/PLD1 ratio PLD1 PLD2 PID2/PLD1 ratio WHYKD36 394 604 1.5 1332 228411To compare the two isoforms of phospholipase D, samples were treatedwith either ML298, a PLD2 inhibitor (355 nM) that would yield PLD1activity or a PLD1 inhibitor (VU0150669, 50 nM) to yield only PLD2activity.

Example 5 Detection and Results of WHYKD Compounds

A photodiode array (PDA) was used to detect WHYKD8 in mouse brain (FIG.1). The sample was readily detected with a discrete peak based on time(left) and with a measurable area under the curve (AUC) (inset).

LC3-II levels were measured in primary cortical neurons following 6hours of treatment with WHYKD1, WHYKD5, or WHYKD1 BafA1 (FIG. 2). Thepresence of LC3-II is an indication of autophagy.

LC3-II levels were then measured in organotypic slice cultures following6 hours of treatment with WHYKD1 (FIG. 3, top panel). Other compounds inthe WHYKD series produced similar results (FIG. 3, bottom panel). RFP isa tag on the tau protein and also can be probed.

These experiments show that the WHYKD series of compounds can induceautophagy and reduce the aggregated forms of tau as well as itsaggresome surrogate p62.

PLD activation converts phospholipids to phosphatidylethanols in thepresence of ethanol. This conversion was measured to show that the WHYKDseries of compounds activate PLD at 10 μM concentration (FIG. 4) and at1 μM (FIG. 5). FIPI is a non-competitive inhibitor of PLD activity andwas used as a negative control.

All patents, patent applications, and publications cited above areincorporated herein by reference in their entirety as if recited in fullherein.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention and all suchmodifications are intended to be included within the scope of thefollowing claims.

1-37. (canceled)
 38. A compound selected from:

or a pharmaceutically acceptable salt, enantiomer, racemate, mixturethereof, or combination thereof.
 39. A pharmaceutical compositioncomprising a compound of claim 1, or a pharmaceutically acceptable saltthereof.
 40. A method of treating a neurodegenerative disease comprisingadministering to a subject in need thereof an effective amount of acompound of claim 1, or a pharmaceutical composition of claim
 39. 41.The method of claim 40, wherein the neurodegenerative disease is atauopathy.
 42. The method of claim 40, wherein the neurodegenerativedisease is Alzheimer's disease.
 43. A method of enhancing autophagicflux comprising providing to a cell or a protein aggregate an effectiveamount of a compound of claim 1, or pharmaceutical composition of claim39.
 44. A method of treating a proteinopathy comprising administering toa subject in need thereof an effective amount of a compound of claim 1,or pharmaceutical composition of claim
 39. 45. A method of treating atauopathy comprising administering to a subject in need thereof aneffective amount of a compound of claim 1, or pharmaceutical compositionof claim 39.